CACTFTPPCA1 (YACT)、Dof (AAAG)、MYB可能参与甘蓝型油菜对氮胁迫的响应

  【目的】  油菜需氮量高但氮素利用率低，氮素源库分配效率被认为是调控植物氮素利用效率的关键因子。在拟南芥中，NRT1.7基因介导了植物韧皮部硝酸盐由衰老叶片向幼嫩叶片和角果中的再转运过程。通过分析鉴定油菜中的NRT1.7基因及其对供氮水平的响应，为进一步系统研究NRT1.7基因提供参考依据。  【方法】  以AtNRT1.7基因序列为基础序列，采用生物信息学方法鉴定了白菜、甘蓝和甘蓝型油菜中NRT1.7的同源基因，预测和分析了该基因拷贝数、系统进化、进化选择压力、分子特征、保守基序、跨膜结构域、染色体定位、基因结构及其启动子区域所能结合的顺式作用元件，同时采用荧光定量PCR分析了甘蓝型油菜BnaNRT1.7s的组织表达模式及其对氮胁迫的响应。氮素响应试验以甘蓝型油菜幼苗为材料，在NO₃?-N 9.0 mmol/L溶液中培养10天后，直接测定NRT1.7基因表达量；转入NO₃?-N 0.3 mmol/L 溶液中 (低氮胁迫) 或在无氮溶液中饥饿处理3天后，恢复NO₃?-N 9.0 mmol/L 溶液培养，再测定NRT1.7基因表达量。  【结果】  甘蓝型油菜NRT1.7s家族包含6个成员，系统进化分析表明BnaNRT1.7s与拟南芥进化相似，分布在相近的分支。BnaNRT1.7s家族所有基因成员的Ka/Ks值均小于1.0，受到强烈的纯化选择作用。BnaNRT1.7s家族所有基因成员均属于稳定的两性蛋白，含12～13个跨膜结构域。基因结构相似，均含有3个内含子，且CACTFTPPCA1 (YACT)、Dof (AAAG)、MYB是启动子上丰度较大的顺式作用原件，可能参与了植物对氮素的响应。实时荧光定量PCR结果表明，甘蓝型油菜中NRT1.7基因会受到不同氮素水平的调控。长期 (72 h) 低氮处理，根部BnaA7.NRT1.7b和BnaC6.NRT1.7b基因的表达上调而抑制地上部BnaCn.NRT1.7基因的表达，共同调控植物对低氮胁迫的适应能力。氮饥饿3天后供氮6 h，地上部和根部BnaNRT1.7的基因表达均受到抑制。基因共表达网络分析显示，低氮胁迫下，BnaCn.NRT1.7和BnaC6.NRT1.7b基因分别在地上部和根部氮素再分配中起主导作用。  【结论】  甘蓝型油菜NRT1.7蛋白进化过程相对保守，基因结构相似，启动子上的顺式作用原件CACTFTPPCA1 (YACT)、Dof (AAAG)、MYB可能参与了甘蓝型油菜对氮胁迫的响应。

CACTFTPPCA1 (YACT), Dof (AAAG) and MYB may be involved in the molecular response of Brassica napus to nitrogen stress

  【Objectives】  Oilseed rape requires high N supply whereas exhibits low N use efficiency (NUE). The allocation of N source and sink is considered the key factor controlling the NUE of plants. NRT1.7 gene in Arabidopsis mediates the re-transport process of phloem nitrates from senescent leaves to young leaves and horns. We identified and analyzed the expression of NRT1.7 gene in rapeseed, and studied their response to N levels.  【Methods】  NRT1.7 homologous genes in Brassica rapa, Brassica oleracea and Brassica napus were identified by bioinformatics with the basic sequences of AtNRT1.7, and were employed to predict and analyze some bioinformatical parameters of BnaNRT1.7s, including copy number, phylogenetic analysis, evolutionary selection pressure, molecular characteristics, conserved motifs, transmembrane domains, chromosome location, gene structures and the cis-acting elements binged in the promoter regions. Moreover, the expression pattern and gene expression response to nitrogen of BnaNRT1.7s were analyzed with the Real-time quantitative PCR detecting system (qRT-PCR). Seedlings of Brassica napus were used as materials in the N hydroponic experiment. The expression of NRT1.7 gene was determined after cultured in solution of NO₃?-N 9.0 mmol/L for 10 days; then transferred into solution of NO₃?-N 0.3 mmol/L for another 3 days (low-N stress), or into no N solution for another 3 days and then resupplied with NO₃?-N of 9.0 mmol/L for 6 h (N-starvation treatment).  【Results】  The identified BnaNRT1.7 gene family contained 6 members, all of them were in similar innovation process and distributed in the similar branch as Arabidopsis thaliana. The Ka/Ks of BnaNRT1.7s were lower than 0.3, indicating a strong purify selection was acting on the BnaNRT1.7s. All BnaNRT1.7s were stable and amphiphilic protein, which contained 12?13 transmembrane domains. The gene structures of BnaNRT1.7s were stable, which were disrupted by 3 introns. CACTFTPPCA1 (YACT), Dof (AAAG) and MYB were the most abundant cis-acting elements binding to the promoter regions of BnaNRT1.7s, which might be involved in the molecular response of plants to N nutrition. The qRT-PCR showed that the expression of NRT1.7 genes was regulated by different N levels. The expression of BnaA7.NRT1.7b and BnaC6.NRT1.7b genes in roots were up-regulated while BnaCn.NRT1.7 gene in shoots was inhibited by 72 h of N stress, which jointly regulated the rapeseed low-N tolerance. In N starvation treatment, the expression of BnaNRT1.7 genes were inhibited in both the shoots and roots. Geneco-expression network identified that BnaCn.NRT1.7 and BnaC6.NRT1.7b were the central members, which were proposed to play core roles in the phloem N remobilization of both the shoots and roots under limited N stresses, respectively.  【Conclusions】  The evolution of NRT1.7 proteins in oilseed rape are relatively conservative with stable gene structures, CACTFTPPCA1(YACT), Dof (AAAG) and MYB are bound to the promoter regions of BnaNRT1.7s, which may be involved in the molecular response of plants to N stress.